1. Field of the Invention
The present invention relates to the field of separation and isolation or purification of nucleic acids from complex biological or clinical specimens.
2. Brief Description of the Background Art
Various methods of separating deoxyribonucleic acids (DNA) from liquid biological samples are known in the art, but are very time consuming or otherwise plagued by complication.
It is known that DNA adheres to nitrocellulose. The liquid sample containing DNA is applied to a nitrocellulose filter and the DNA adheres or binds to the filter. The problem encountered is that proteins also bind to the nitrocellulose. Therefore this method is not specific for DNA alone.
Another method of separating DNA from samples is ultracentrifugation with sucrose or cesium chloride density gradients. The DNA is separated from other macromolecules such as proteins by this method according to its buoyant density or sedimentation coefficient. The biological sample is layered onto the density gradient in a centrifuge tube and is spun at very high speeds for long periods of time for the DNA to travel through the density gradient. This method, although satisfactory, is very time consuming and labor intensive. The centrifugation time may be 20 hours or more per sample. Furthermore, if the sample is spun too long, the DNA will separate from the sample but will pass entirely through the gradient to the very bottom of the centrifuge tube along with other constituents in the sample. Therefore, this method is also not suitable as a fast and easy method for separating DNA from complex samples.
Chemical methods of separating DNA from liquid samples are also known. Phenol extraction and ethanol precipitation are standard laboratory procedures, but each has its difficulties. Phenol is a toxic substance, and phenol extraction requires subsequent time-consuming procedures such as extraction with other organic reagents or dialysis to purify the sample. Furthermore, the separation of DNA from complex mixtures is subject to interference from molecules with nucleic acid binding properties. Ethanol causes many proteins, not just DNA, to precipitate, so the DNA must then still be separated from all other proteins in the sample.
Finally, agarose or polyacrylamide gel electrophoresis is used to separate DNA from biological samples. In this method the sample is applied to one end of a glass or plastic receptacle containing the gel and an electric current is applied across the length of the receptacle. The negatively charged nucleic acid molecules move toward the anode, the larger molecules moving more slowly. The rates of migration of the molecules depend on their molecular weights and on the concentration and degree of cross linking in the gel material. The DNA is then removed from the gel by cutting out that portion of the gel in which the DNA is located and finally extracting the DNA. Again, this method is time consuming and labor intensive, and the DNA must still be separated from the gel.
When DNA is separated by the electrophoresis gel method, it is necessary for the DNA to be stained in some manner to be visualized. Typically, ethidium bromide (EtBr) is used as the staining agent. Ethidium bromide adheres to the DNA by intercalation between the base pairs of the double helix structure of the DNA. The use of ethidium bromide in staining DNA is described in Sharp, P.A., et al. "Detection of Two Restriction Endonuclease Activities in Haemophilus Parainfluenzae Using Analytical Agarose-Ethidium Bromide Electrophoresis," Biochemistry 12:3055-3063 (1973). This reference discloses a rapid assay for restriction enzymes in which ethidium bromide is used to stain the DNA.
In a somewhat different application of ethidium bromide as a staining agent, ethidium bromide has been linked to a solid support. U.S. Pat. No. 4,119,521, issued to Chirikjian on Oct. 10, 1978, discloses a fluorescent DNA intercalating agent derivative of activated polysaccharides. The derivatives in the patent function as fluorescent stains to provide direct visualization of the DNA and their fractions, under the excitation of short-wave, ultraviolet radiation. The intercalating agents used in the patent are ethidium halides with the preferred agent being ethidium bromide. This agent is coupled covalently to an activated polysaccharide such as agarose.
Dervan P. B., and Becker, M. M., "Molecular Recognition of DNA by Small Molecules. Synthesis of Bis(methidium)spermine, a DNA Polyintercalating Molecule," J. Amer. Chemical Society 100:1968-1970 (1978), discloses the synthesis and study of bis(methidium)spermine (BMSp). This molecule was described as a polyintercalator due to the presence of two separate methidium intercalating agents connected by means of the spermine linker. Spermine was chosen to link the intercalators because of its known affinity for nucleic acid and its length, which allows a geometry sufficient to reach nonadjacent intercalation sites in accordance with the neighbour exclusion binding model.
The studies of Dervan and Becker further showed that BMSp and ethidium bromide intercalated in similar manners. However, the BMSp has a binding constant several orders of magnitude stronger than the monomer, ethidium bromide.
Vachek, A. T. et al., Analytical Biochemistry 124:4-420 (1982), discloses an ethidium-acrylamide affinity medium for recovering nucleic acids from free solution and from polyacrylamide and agarose gels. This affinity medium is composed of an acrylamide matrix to which ethidium bromide is attached. Apparently, the nucleic acids can be eluted from this medium with a buffered salt solution and directly concentrated by ethanol precipitation.
The ethidium-acrylamide affinity medium is synthesized by the reaction, in the presence of a polyacrylamide matrix, of ethidium bromide, BIS (N,N'-methylenebisacrylamide), TEMED(N,N,N',N'-tetramethylethylenediamine), and ammonium persulfate in a suitable buffer. When ethidium bromide is present, it presumably becomes covalently linked to the acrylamide matrix through methylenebisacrylamide spacer arms.
This reference discloses the use of Bio-Gel P-4 as the particulate acrylamide matrix. The cross-linking properties of methylenebisacrylamide are evidently important for the linking of ethidium to Bio-Gel P-4. The binding of ethidium to this gel is also somewhat dependent on the composition of the buffer in the reaction. The authors noted that allowing ample time for interaction with the affinity medium (i.e., flow rates of 0.44 ml/min for a 3-ml bed volume in a 6-ml syringe and a 15-min equilibration) was essential for quantitative binding. However, elution of the RNA shortly after 15-min equilibration with the column was necessary to avoid some apparently irreversible binding.
Thomas, K. A. and A. N. Schechter, Analytical Biochemistry, 91:209-223 (1978) discloses direct physical measurements on substituted agarose gels and evidence of intercalation of gel-bound ethidium into transfer-RNA. This reference reports that the cation of the salt ethidium bromide (3,8- diamino-5-ethyl-6-phenylphenanthridinium bromide) has been covalently linked to an agarose matrix through an intermediate 3,3'-diaminodipropylaminosuccinyl spacer arm.
The paper also discloses the stoichiometry and nature of the binding of tRNA to ethidium cations that have been covalently bound to an agarose gel as determined from partition binding and direct spectral measurements of the gel itself. Various measurements were made to study the ethidium-tRNA interaction. Fluorescence enhancement and spectral red shift were observed and determined to be proportional to the amount of tRNA bound to the gel. Experiments also showed that increasing the NaCl concentration decreased the percentage of tRNA bound to the gel. A molar concentration of approximately 1.3 or higher resulted in 0% binding. The authors concluded that the ethidium bromide bound to the tRNA by intercalation. This reference does not document any actual or speculative use of the capture reagent for isolating DNA or RNA from complex unpurified mixtures such as clinical samples. Therefore, a need continues to exist for a rapid and efficient method of separating DNA from unpurified clinical samples.